The present invention relates to novel compounds and to novel complexes of these compounds with metals. The novel compounds and complexes of the present invention find utility in diagnostic and therapeutic methods.
Metal complexes, such as those containing radioactive metals, are finding increasing use as diagnostic and therapeutic agents. Of particular interest are those complexes containing bioactive moieties capable of being selectively taken up at a desired site to facilitate evaluation or treatment of a subject.
The present invention addresses the need in the art for such complexes and the ligands from which they are prepared.
The present invention provides novel compounds, also referred to herein as ligands, of the following formula I: 
where
Q is the group xe2x80x94(C(RR))m1xe2x80x94(Y1)nxe2x80x94(C(RR))m2xe2x80x94(Y2xe2x80x94(C(RR))m3)n1xe2x80x94, Y1 and Y2 are each independently xe2x80x94NRxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94 or xe2x80x94Sexe2x80x94; n and n1 are each independently 0 or 1; and m1, m2 and m3 are each independently 0 or an integer from 1 to 4; provided that m1 and m2 are not both 0, that m1+m2+n+n1 is less than 6 and that a carbon atom bearing an R group is not directly bonded to more than one heteroatom;
each R and R* group is independently:
(i) R1;
(ii) alkoxy;
(iii) hydroxy;
(iv) halogen, especially fluoro;
(v) haloalkyl;
(vi) xe2x80x94OR1;
(vii) xe2x80x94C(O)xe2x80x94OR1;
(viii) xe2x80x94C(O)xe2x80x94N(R1)2;
(ix) xe2x80x94N(R1)2xe2x80x94;
(x) xe2x80x94N(R1)xe2x80x94COR1;
(xi) -alkyl-C(O)xe2x80x94OR1;
(xii) -alkyl-C(O)xe2x80x94N(R1)2;
(xiii) -alkyl-N(R1)2xe2x80x94;
(xiv) -alkyl-N(R1)xe2x80x94COR1;
(xv) -aryl-C(O)xe2x80x94OR1;
(xvi) -aryl-C(O)xe2x80x94N(R1)2;
(xvii) -aryl-N(R1)2xe2x80x94;
(xviii) -aryl-N(R1)xe2x80x94COR1;
(xix) nitrile;
(xx) acyl;
(xxi) acyloxy;
(xxii) heterocyclo;
(xxiii) hydroxyalkyl;
(xxiv) alkoxyalkyl;
(xxv) hydroxyaryl;
(xxvi) arylalkyl;
(xxvii) xe2x80x94SO2xe2x80x94R1;
(xxviii) -alkyl-SO2xe2x80x94R1;
(xxix) xe2x80x94(A)pxe2x80x94Ra, where A is a linking group, p is 0 or a positive integer, and Ra is a bioactive moiety or a reactive group capable of forming a covalent bond to a bioactive moiety; or
(xxx) two of the groups, taken together with one or more atoms to which they are bonded, form a saturated or unsaturated, spiro or fused, carbocyclic (such as fused 1,2-phenyl) or heterocyclic ring which may be unsubstituted or substituted by one or more groups selected from the groups (i) to (xxix) above;
each R1 is independently hydrogen, alkyl, alkenyl, alkynyl or aryl; and
G1 and G2 are each independently xe2x80x94OH or xe2x80x94N(R2)2, provided that at least one of G1 or G2 is xe2x80x94N(R2)2, where each R2 is independently hydrogen, alkyl, aryl, acyl or xe2x80x94(A)pxe2x80x94Ra.
The present invention also provides complexes of the aforementioned compounds of the formula I with metals, preferably rhenium or technetium, and the use of these complexes in diagnostic and therapeutic methods. Further provided by the present invention are kits for preparing the metal complexes of the present invention.
In certain embodiments, the present invention provides complexes, without a specific bioactive moiety, that localize selectively in an organ or organ system and may be useful for heart, brain, hepatobiliary or other organ imaging. In other embodiments, the present invention provides complexes containing bioactive moieties, such as receptor or hypoxia-localizing moieties, which retain the biochemical behavior and affinity of the free moieties and which are capable of rapidly providing increased amounts of a desired radionuclide selectively to targeted areas. Certain complexes of the invention may be labeled at ambient temperature with suitable, easy-to-use radionuclides; and some complexes may be membrane permeable, allowing intracellular delivery.
The present invention is described further as follows.
Definitions
Listed below are definitions of terms used to describe the present invention. These definitions apply to the terms as they are used throughout the specification unless otherwise indicated.
The terms xe2x80x9calkylxe2x80x9d or xe2x80x9calkxe2x80x9d, as used herein alone or as part of another group, denote optionally substituted, straight and branched chain saturated hydrocarbon groups, preferably having 1 to 12 carbons in the normal chain. Most preferably, these groups are lower alkyl groups. Exemplary unsubstituted alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl and the like. Exemplary substituents include one or more of the following groups: halo, alkoxy, alkylthio, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, carboxyl (xe2x80x94COOH), amino, alkylamino, dialkylamino, formyl, alkylcarbonyloxy, alkylcarbonyl, heterocyclo or thiol (xe2x80x94SH). Preferred alkyl groups are unsubstituted alkyl, haloalkyl, arylalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, aryloxyalkyl and hydroxyalkyl groups.
The terms xe2x80x9clower alkylxe2x80x9d or xe2x80x9clower alkxe2x80x9d, as used herein, denote optionally substituted groups as described above for alkyl but having only 1 to 4 carbon atoms in the normal chain.
The terms xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkylthioxe2x80x9d denote an alkyl group as described above bonded through an oxygen linkage (xe2x80x94Oxe2x80x94) or a sulfur linkage (xe2x80x94Sxe2x80x94), respectively. The term xe2x80x9calkylcarbonylxe2x80x9d, as used herein, denotes an alkyl group bonded through a carbonyl group. The term xe2x80x9casalkylcarbonyloxyxe2x80x9d, as used herein, denotes an alkyl group bonded through a carbonyl group which is, in turn, bonded through an oxygen linkage.
The term xe2x80x9calkenylxe2x80x9d, as used herein alone or as part of another group, denotes optionally substituted, straight and branched chain hydrocarbon groups containing at least one carbon to carbon double bond in the chain, and preferably having 2 to 10 carbons in the normal chain. Exemplary unsubstituted alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl and the like. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The term xe2x80x9calkynylxe2x80x9d, as used herein alone or as part of another group, denotes optionally substituted, straight and branched chain hydrocarbon groups containing at least one carbon to carbon triple bond in the chain, and preferably having 2 to 10 carbons in the normal chain. Exemplary unsubstituted alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl and the like. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The term xe2x80x9ccycloalkylxe2x80x9d, as used herein alone or as part of another group, denotes optionally substituted, saturated cyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring. Exemplary unsubstituted cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl and adamantyl. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The term xe2x80x9ccycloalkenylxe2x80x9d, as used herein alone or as part of another group, denotes such optionally substituted groups as described above for cycloalkyl, further containing at least one carbon to carbon double bond forming a partially unsaturated ring.
The term xe2x80x9carylxe2x80x9d, as used herein alone or as part of another group, denote optionally substituted, homocyclic aromatic groups, preferably containing 1 or 2 rings and 6 to 12 ring carbons. Exemplary unsubstituted aryl groups include phenyl, biphenyl and naphthyl. Exemplary substituents include one or more, preferably three or fewer, nitro groups, alkyl groups as described above and/or groups described above as alkyl substituents. Preferred aryl groups are unsubstituted aryl and hydroxyaryl.
The term xe2x80x9ccarbocyclicxe2x80x9d, as used herein alone or as part of another group, denotes optionally substituted saturated, partially unsaturated or aromatic homocyclic hydrocarbon ring systems such as the cycloalkyl, cycloalkenyl or aryl groups described above.
The terms xe2x80x9cheterocycloxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d, as used herein alone or as part of another group, denote optionally substituted fully saturated or unsaturated, aromatic or non-aromatic cyclic groups having at least one heteroatom in at least one ring, preferably monocyclic or bicyclic groups having 5 or 6 atoms in each ring. The heterocyclic group may, for example, have 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring. Each heterocyclic group may be bonded through any carbon or heteroatom of the ring system. Preferred groups include those of the following formula, which may be bonded through any atom of the ring system: 
where r is 0 or 1 and T is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Nxe2x80x94R3 or xe2x80x94CHxe2x80x94R3 where R3 is hydrogen, alkyl, aryl or arylalkyl. Exemplary heterocyclic groups include the following: thienyl, furyl, pyrrolyl, pyridyl, imidazolyl, pyrrolidinyl, piperidinyl, azepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, morpholinyl, piperazinyl, 4-alkylpiperazinyl, 4-alkylpiperidinyl, 3-alkylpyrrolidinyl, oxazolyl, pyrazolyl, thiophenyl, pyridazinyl, thiazolyl, triazolyl, pyrimidinyl, 1,4-dioxanyl, benzoxadiazolyl and benzofurazanyl. Exemplary substituents include one or more alkyl groups as described above and/or one or more groups described above as alkyl substituents.
The terms xe2x80x9chalogenxe2x80x9d and xe2x80x9chaloxe2x80x9d, as used herein alone or as part of another group, denote chlorine, bromine, fluorine and iodine.
The term xe2x80x9cacylxe2x80x9d, as used herein alone or as part of another group, denotes the moiety formed by removal of the hydroxyl group from the group xe2x80x94COOH of an organic carboxylic acid. Exemplary acyl groups include alkylcarbonyl, arylcarbonyl or carbocyclo- or heterocyclocarbonyl. The term xe2x80x9cacyloxyxe2x80x9d, as used herein alone or as part of another group, denotes an acyl group as described above bonded through an oxygen linkage (xe2x80x94Oxe2x80x94).
For the above optionally substituted groups, reference to a specific substituent may be made without excluding the presence of other substituents. Thus, for example, xe2x80x9chydroxyalkylxe2x80x9d is a straight or branched chain saturated hydrocarbon group bearing at least one hydroxy substituent and, optionally, one or more additional substituents.
The expression xe2x80x9cthiol protecting groupxe2x80x9d, as used herein, denotes a group which may be cleaved from sulfur to yield a thiol group without destruction of the remainder of the molecule.
The expressions xe2x80x9cbioactive groupxe2x80x9d and xe2x80x9cbioactive moietyxe2x80x9d, as used herein, denote a group which is capable of functioning as a metabolic substrate, catalyst or inhibitor, or is capable of being preferentially taken up at a selected site of a subject, such as by possessing an affinity for a specific receptor.
The term xe2x80x9clinking groupxe2x80x9d, as used herein, denotes a group which, alone or together with one or more other groups, covalently bonds a bloactive group to the remainder of a compound of the formula I of the present invention.
The various substituents of the ligands of the present invention may be chosen to form stable compounds.
Compounds of the Formula I
The compounds of the formula I of the present invention may be prepared by methods such as those illustrated in, or analogous to, the following Reaction Schemes and in the Examples herein. 
The above reaction schemes illustrate general methods for the preparation of compounds of the formula I.
According to the Reaction Scheme as shown in reaction A, a compound of the formula II may be contacted with approximately two or more molar equivalents of a haloketone III (where X is halogen) to form the diketone IV. A compound of the formula I may then be prepared in reaction B by conversion of the keto groups of the compound IV to hydrazone groups by methods such as treatment with a substituted hydrazine.
Alternatively, as shown in reaction C, a compound of the formula II may be contacted with an approximately equimolar amount of a compound of the formula V where X is halogen (preferably chloro), preferably in the presence of a tertiary amine such as diisopropylethylamine, to provide a compound of the formula VI. The compound of the formula VI may then be contacted in reaction D with a haloketone III to form a compound of the formula VII. The keto group of the compound of the formula VII may then be converted in reaction E to a hydrazone group by a method such as that described above for reaction B to form a compound of the formula I.
As the compounds of the formula II possess two terminal groups, it may be desirable to protect one of these groups to obtain preferential reaction through the unprotected amino group. Thus, compounds of the formula IIPro may be employed in another alternative method. As shown in reaction F, compounds of the formulae IIPro and III are contacted in an approximately equimolar ratio to form a monoketone of the formula VIII. The keto group of the compound VIII may be converted in reaction G to a hydrazone group by a method such as that described above for reaction B to form a compound of the formula IX. The compound of the formula IX is deprotected in reaction H to form a compound of the formula VI for further reaction. The latter compound may then be contacted with a haloketone of the formula III to yield a compound of the formula VII, and reaction E conducted to yield a compound of the formula I as described above.
(Compounds of the formula III may be obtained by methods such as those described by Pfleiderer et al., Liebigs Ann. Chem., 99, 3008 (1966).)
Additionally, compounds of the formula I may be prepared where any R group can be readily incorporated into the ligand in the last reaction step by reacting a monooxime with a hydrazine that bears the desired R group. This is of particular advantage when the R group is hard to synthesize or is so labile that it might not survive incorporation into the ligand at an earlier step.
In all of the above reactions described for preparing compounds of this invention, groups such as sulfur groups, amine groups and ketone groups may be protected where appropriate during the various reactions, and the so-protected resulting products thereafter deprotected by known techniques.
Preferred Compounds
Compounds of the formula I are preferred where n, n1 and m2 are 0 and therefore Q is (C(RR))m1; R and R* are independently H or alkyl groups, especially unsubstituted lower alkyl groups such as methyl; and at least one R2 is hydrogen.
Particularly preferred compounds are those where Q is (CH2)2 or (CH2)3; the remaining R and R* groups are CH3; G1 is OH or NH2; and G2 is NH2, NHCH3, NHC6H5 or NHC(O)C6H5.
Preferred linking groups, and preferred Ra groups, are discussed below.
Metal Complexes
The compounds of the formula I may be employed as ligands for the formation of metal complexes.
The metal complexes of the present invention may be formed by complexing a compound of the formula I with a radioactive or non-radioactive metal, including a metal having an atomic number 22-31, 39-49 or 73-82, especially rhenium or technetium, preferably under basic conditions.
An exemplary method for the formation of a metal complex of the present invention is that where a complex or salt of the desired metal in the desired oxidation state and containing one or more easily displaceable (i.e., labile) ligands (for example, H2O, halogen (eg., Cl), NO3xe2x88x92, or sugars) is mixed with ligand(s) of the present invention at a pH value suitable for forming the desired complex. The labile ligand is displaced from the metal by the ligand(s) of the present invention to form a metal complex of the present invention.
Illustrations of these methods are shown below:
xe2x80x83(Met)(Liglab)4+(Liginv)xe2x86x92(Met)(Liginv)+4(Liglab)xe2x80x83xe2x80x83(1)
where
Met is a metal in a desired oxidation state;
Liglab is a labile ligand such as H2O, Clxe2x88x92, Brxe2x88x92, Fxe2x88x92 or NO3xe2x88x92; and
Liginv is a ligand of the present invention.
(Met)OCl4xe2x88x92+(Liginv)xe2x86x92(Met)O(Liginv)+4Clxe2x88x92xe2x80x83xe2x80x83(2)
(Met)O2(Ligmono)4+(Liginv)xe2x86x92(Met)O2(Liginv)+4(Ligmono)xe2x80x83xe2x80x83(3)
where Ligmono is a monodentate ligand such as pyridine, halogen, phosphine or amine.
(Met)(Ligbi)2+(Liginv)xe2x86x92(Met)(Liginv)+2(Ligbi)xe2x80x83xe2x80x83(4)
or
(Met)O(Ligbi)2+(Liginv)xe2x86x92(Met)O(Liginv)+2(Ligbi)xe2x80x83xe2x80x83(5)
where Ligbi is a bidentate ligand such as a sugar, a diol, a bisamine, bipyridine or phosphine, and where, for each equation (1) to (5) above, the appropriate charge balance is employed.
Alternatively, the metal complexes of the present invention may be prepared from a metal in an oxidation state different from that of the desired complex. An example of such a method is that where either a reducing agent or an oxidizing agent (depending on the oxidation state of the metal used, and the oxidation state of the desired final product) is added to the reaction mixture containing metal to bring the metal to the desired oxidation state. The oxidant or reductant may optionally be used to form an intermediate complex in the desired oxidation state but with labile ligands which are then displaced by a desired chelating ligand of the present invention; or the oxidant or reductant may be added to the reaction mixture containing metal along with the desired ligand to achieve the change to the desired oxidation state and chelation to the desired metal in a single step.
Exemplary metal complexes of the present invention may be shown as the following formula I complex: 
where the R and R* groups are as defined above, and where M can be a radioactive or non-radioactive metal which may optionally have other ligand(s) W1 and/or W2 in the unfilled coordination sites thereof. A hydrogen bond may be formed between G1 and G2 on complex formation. This will enhance the stability of the complex relative to a non-hydrogen bonded complex. Radioactive metals, for example, technetium, are preferred in these complexes. Preferably, in the cases where M is rhenium or technetium, the 
portion can be shown as 
Other suitable co-ligands, W1 and W2, to form these complexes may include, but are not limited to, mono-, di- or tridentate ligands which, when combined with the ligands I form neutral metal complexes, particularly of technetium or rhenium, with the metal preferably in the +5 oxidation state.
The metal complexes of the present invention find utility as diagnostic and/or therapeutic agents. Thus, the present invention provides methods for the diagnosis of the presence and/or status of a disease state, or for the treatment of a disease state, comprising the step of administering a metal complex of the present invention to a subject in need thereof. The metal complexes of the present invention may be administered by any appropriate route such as orally, parenterally (for example, intravenously, intraperitoneally, intramuscularly or subcutaneously), or by any other suitable method. For example, the complexes of this invention may be administered to a subject by bolus or slow infusion intravenous injection.
The amount administered may be selected based on the desired use, such as to produce a diagnostic image of an organ or other site of a subject or a desired radiotherapeutic effect, by methods known in the art. Exemplary dosages are those employing about 30-200 mCi rhenium (for radiotherapy) or about 10-60 mCi technetium (for imaging). The xe2x80x9csubjectxe2x80x9d of the methods of the present invention is preferably a mammal such as a domestic mammal, for example, a dog, cat, horse or the like, or most preferably, a human. Depending upon the metal and ligand used, the complexes of the present invention may be employed as, for example, imaging agents useful for imaging organs such as the heart or brain (where the complex may cross the blood-brain barrier), or for imaging the hepatobiliary system. The complexes of the invention are especially useful when attached to a biolocalizing moiety, for example, for the imaging of hypoxic tissue, and as therapeutic agents, such as hypoxic tissue cytotoxins, or radiosensitizers.
Metal complexes which have a permeability through cell membranes greater than that of 14C-sucrose, such as those containing a hypoxia-localizing moiety, are one-embodiment of the diagnostic or therapeutic methods of the present invention. Cell permeability is a property of a cell membrane which describes the mobility of extraneous molecules (permeants) within the internal structure of the membrane (Stein, xe2x80x9cTransport and Diffusion Across Cell Membranexe2x80x9d, New York Academic Press Inc. (1986); Kotyk et al., Biophysical Chemistry of Membrane Functions, Chichester, UK: John Wiley and Sons (1988)). Molecules to which the membrane is permeable are able to penetrate through the membrane to reach the environment on the opposite side. Cell permeability may be determined by methods such as those described in U.S. patent application Ser. No. 08/054,120, filed Apr. 27, 1993 by Linder et al., incorporated herein by reference.
Preferred complexes of the present invention are those comprising a compound of the formula I complexed with a radionuclide such as technetium or rhenium.
Rhenium is useful as a radiotherapy agent. The rhenium employed is generally one of the radionuclides Re-186 or Re-188, or a mixture thereof, which mixture may also include Re-185 and/or Re-187. Preparation of the complexes of the present invention where the metal is rhenium may be accomplished using rhenium in the +5 or +7 oxidation state. Examples of compounds in which rhenium is in the Re(VII) state are NH4ReO4 or KReO4. Re(V) is available as, for example, [ReOCl4](NBu4), [ReOCl4](AsPh4), ReOCl3(PPh3)2 and as ReO2(pyridine)4+. (Ph is phenyl; Bu is n-butyl). Other rhenium reagents, such as xe2x80x9ccarrier rheniumxe2x80x9d, capable of forming a rhenium complex may also be used. The phrase xe2x80x9ccarrier rheniumxe2x80x9d means that the rhenium compounds used contain non-radioactive rhenium at concentrations  greater than 10xe2x88x927M.
Technetium is particularly useful as a diagnostic imaging agent. The technetium employed is preferably one or more of the radionuclides 99mTc, 94mTc or 96mTc. The preferred radioisotope for medical imaging is 99mTc. Its 140 keV xcex3-photon is ideal for use with widely-available gamma cameras. It has a short (6 hour) half life, which is desirable when considering patient dosimetry. 99mTc is readily available at relatively low cost through commercially-produced 99Mo/99mMTc generator systems. Preparation of the complexes of this invention where the metal is technetium may be accomplished using technetium in the form of the pertechnetate ion. For 99mTc, the pertechnetate ion is preferably obtained from commercially available technetium-99m parent-daughter generators; such technetium is in the +7 oxidation state. The generation of the pertechnetate ion using this type of generator is well known in the art, and is described in more detail in U.S. Pat. Nos. 3,369,121 and 3,920,995. These generators may generally be eluted with saline solution, and the pertechnetate ion obtained as the sodium salt. Pertechnetate may also-be prepared from cyclotron-produced radioactive technetium using procedures well known in the art.
The formation of a technetium complex is preferably achieved by mixing pertechnetate ion in normal saline with the appropriate ligand. An appropriate buffer or physiologically acceptable acid or base may be used to adjust the pH to a value suitable for labeling the ligand. This appropriate value of pH will vary depending upon the nature of the ligand; for example, for ligands of the formula I, a pH in the range between xcx9c5.5 to xcx9c9.5 is suitable, preferably a pH value in the range of 7.0 to 8.5. A source of reducing agent may then be added to bring the pertechnetate down to the oxidation state of Tc(V) for chelation with the ligand. Stannous ion is the preferred reducing agent, and may be introduced in the form of a stannous salt such as stannous chloride, stannous fluoride, stannous tartrate, stannous diethylenetriamine pentaacetic acid or stannous citrate, or the like. The reaction is preferably run in an aqueous or aqueous/alcohol mixture, at or about room temperature, using a reaction time of about 1 minute to about 1 hour. The reducing agent is preferably present at a concentration of 5 to 50 xcexcg/mL. The ligand is preferably present in a concentration of 0.5 to 2 mg/mL. Optionally, co-ligands W1 and W2 discussed above may be added.
Alternatively, the technetium complexes of this invention may be prepared by ligand exchange. A labile Tc(V) complex may be prepared by the reduction of TcO4xe2x88x92 in the presence of a ligand which forms a labile technetium complex, such as ethylene glycol, mannitol, or the hydroxy-carboxylate ligands glucoheptonate, gluconate, citrate, malate or tartrate, at a pH value which is appropriate for the exchange ligand employed (usually 5 to 8). A reducing agent, such as the stannous salts described above, may be added, causing the formation of a labile reduced complex of Tc with the exchange ligand. This reduced Tc complex is then mixed with the ligand ultimately desired, at an appropriate pH value (as described above). The labile exchange ligand is displaced from the metal by the desired ligand, thus forming the technetium complexes of this invention.
Metal complexes of the present invention are preferred in which a compound described above under the section entitled xe2x80x9cPreferred Compoundsxe2x80x9d is complexed with a metal, most preferably, with rhenium or technetium. Ligands which form single, neutral complexes are preferred. Exemplary complexes include those having the following structures: 
where M1 is technetium.
While metal complexes of the present invention containing one or more bioactive groups as described below are useful, those complexes of the present invention lacking such groups are also useful, for example, in flow or organ imaging. Preferred complexes lacking bioactive group(s) are those produced from lipophilic ligands suitable for imaging the heart, brain or hepatobiliary system, or anionic or cationic groups suitable for imaging renal or hepatic function.
Bioactive Groups
A bioactive group (Ra) of the compounds of the present invention is capable of functioning as a metabolic substrate, catalyst or inhibitor, for example, to aid in clearance of the complex from non-target tissue; or is capable of being preferentially taken up at a selected site of a subject, such as by possessing an affinity for a cellular recognition site such as a receptor, enzyme or transport mechanism; or contain reactive groups for coupling to proteins; or effects tissue localization by another biochemical process. Thus, complexes of the present invention are contemplated where one or more bioactive groups are bound to the remainder of the complex which retain their desired bioactivity when so bound.
Exemplary bioactive groups include amphetamines, barbiturates, sulfonamides, monoamine oxidase substrates and inhibitors, hormones, enzymes, lipids, ligands for cell membrane receptors, antihypertensives, neurotransmitters, amino acids and oligopeptides, radiosensitizers, steroids (such as estrogen or estradiol), monoclonal or polyclonal antibodies or fragments thereof, carbohydrates (such as glucose derivatives), fatty acids, substrates for muscarinic receptors (such as 3-quinuclidinyl benzilate), substrates for dopamine receptors (such as spiperone), biotin, peptides and proteins which bind to specific receptors, substrates for benzodiazepine receptors and hypoxia-localizing moieties.
Complexes of the present invention containing bioactive groups are useful in that they employ properties, e.g., receptor binding, metabolism, etc., of a particular biochemically active group to provide imaging or treatment of a particular site or function. These complexes, especially where the metal is 99mTc, provide highly effective, relatively easy to use diagnostic imaging products which are characterized by a covalent bond between the radionuclide complex and the bioactive group while substantially retaining the uptake properties of the free bioactive group. Examples of diagnostic uses for the complexes of the present invention include, but are not limited to, imaging of hypoxic tissue, e.g., in the heart, brain, lungs or in tumors; imaging of the brain and lungs when the bioactive group is a lipophilic amine-containing compound, e.g., an amphetamine; imaging of the brain, heart or tumors when the bioactive group is a sugar (e.g., a glucose derivative); imaging of the heart when the bioactive group is a fatty acid; imaging of steroid receptor sites when the bioactive group is a steroid (e.g., an estrogen for imaging breast carcinoma); and imaging of sites of infection when the bioactive group is a chemotactic peptide with affinity for blood cell types which localize at the site of infection.
In addition to diagnostic agents, the present invention also provides stably bound complexes for radiotherapeutic indications such as those indications described in U.S. Pat. No. 4,871,836. For example, Re complexes of the present invention which include estradiols can be used in the treatment of breast carcinoma. Also, to the extent that hypoxic tissue is known to be present in tumors, Re complexes of the present invention where the bioactive group is a hypoxia-localizing moiety are suitable for radiotherapy. The complexes of this invention where the metal is Re, for use in radiotherapy, are preferably injected into humans and allowed to concentrate at the desired site. Targeting of the radionuclide to a desired site with great specificity may thus be achieved. Radiotherapy is contemplated for those areas where a sufficient quantity of interacting sites (for example, estrogen receptors or hypoxic tissue) are present so as to provide therapeutic levels of the radionuclide to the area needing treatment.
When the bioactive group Ra is a steroid, it is understood that either a steroid, a substituted steroid derivative or a non-steroidal derivative. may be employed provided that the Ra group chosen has an affinity for the steroid receptor. For example, Ra may be the steroid estradiol: 
The estradiol group may be bonded to the remainder of the complex at any available position on the molecule, but is preferably bonded through a linking group to either an atom in the B ring or an atom in the D ring. Additionally, the estradiol molecule may be substituted at available positions by one or more R groups where R is as defined above. Alternatively, the steroid molecule may be replaced by a non-steroidal diol with a known affinity for the estrogen receptor, such as 
where (A)p and R are as defined above.
When the bioactive group is a substrate for a muscarinic receptor, the xe2x80x94(A)pxe2x80x94Ra portion of the complex is preferably the group: 
where (A)p and R are as defined above and R4 is a tertiary or quaternary amine, such as 3-quinuclidinol or a substituted 3-quinuclidinol, or the following compounds: 
Linking Groups
The linking group(s) (A)p of the compounds of the present invention, when present (that is, when p is greater than zero), may be any one or more moieties which can serve to distance physically, or otherwise isolate, the bioactive group from the remainder of the compound of the formula I or complex thereof. The presence of such linking group(s) may be desirable, for example, where a bioactive group may be inhibited in its action by the remainder of the complex. In considering the various linking groups which may be employed, it is understood that p may be any convenient value depending upon the design choice for the desired complex. Preferably, p is xe2x89xa620 and is most preferably xe2x89xa610.
Examples of linking groups which may be employed alone (where p is one), or together to form a straight or branched chain (where p is greater than one) and which may be bonded to the remainder of the ligand from either end are: xe2x80x94CH2xe2x80x94, xe2x80x94CHR5xe2x80x94, xe2x80x94CR5R6xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90CR5xe2x80x94, xe2x80x94CR5xe2x95x90CR6xe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, cycloalkyl, cycloalkenyl, aryl (e.g., p-phenylene or hydroxy substituted p-phenylene), heterocycle, oxygen, sulfur, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94HCxe2x95x90Nxe2x80x94, xe2x80x94CR5xe2x95x90Nxe2x80x94, xe2x80x94NR5xe2x80x94 and xe2x80x94CSxe2x80x94; wherein R5 and R6 are independently selected from alkyl, alkenyl, alkoxy, aryl, 5- or 6-membered nitrogen- or oxygen-containing heterocycles, halogen, hydroxy or hydroxyalkyl.
When present, the preferred values for (A)p (bonded to the remainder of the ligand from either end) are alkyl, oxa-alkyl, hydroxyalkyl, hydroxyalkoxy, alkenyl, arylalkyl, arylalkylamide, alkylamide, alkylamine and (alkylamine)alkyl.
The most preferred values for (A)p are selected from the following (bonded to the remainder of the ligand from either end): xe2x80x94(CH2)1-5xe2x80x94 (especially methyl or ethyl), xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CH2xe2x80x94, xe2x80x94(CH2)1-2xe2x80x94C(O)xe2x80x94NHxe2x80x94(CH2)1-3xe2x80x94, xe2x80x94C6H5xe2x80x94(CH2)1-2xe2x80x94, xe2x80x94(CH2)1-2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94, xe2x80x94(CH2)2xe2x80x94Oxe2x80x94, xe2x80x94CH2CH(OH)CH2OCH2xe2x80x94, xe2x80x94CH2xe2x80x94C(O)xe2x80x94NHxe2x80x94CHxe2x80x94C6H5xe2x80x94, xe2x80x94(Axe2x80x2xe2x80x94Oxe2x80x94Axe2x80x3)1-3, and xe2x80x94(Axe2x80x2xe2x80x94NHxe2x80x94Axe2x80x3)1-3; where Axe2x80x2 and Axe2x80x3 are the same or different alkyl or aryl groups and C6H5 is p-phenylene.
Kits for Forming Metal Complexes
It is convenient to prepare the complexes of the present invention at, or near, the site where they are to be used. A single, or multi-vial kit that contains all of the components needed to prepare the complexes of this invention, other than the radionuclide ion itself, is an integral part of this invention.
A preferred single-vial kit of the present invention comprises a ligand of the formula I and a source of a pharmaceutically acceptable reducing agent such as a stannous salt. Most preferably, in addition, the kit is buffered with a pharmaceutically acceptable acid or base to adjust the pH to a desired value for complex formation as described above. It is preferred that the kit contents be in lyophilized form. Such a single vial kit may optionally contain exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and may also contain reaction modifiers, such as diethylenetriaminepentaacetic acid or ethylenediamine tetraacetic acid. Additional additives, such as solubilizers (for example xcex1-, xcex2- or xcex3-cyclodextrin), antioxidants (for example ascorbic acid), fillers (for example, sodium chloride) may be employed to improve the radiochemical purity and stability of the final product, or to aid in the production of the kit.
A preferred multi-vial kit of the present invention comprises, in one vial, the components, other than the radionuclide itself, required to form a labile radionuclide (especially Tc(V)) complex as described above, that is, an exchange ligand and a pharmaceutically acceptable reducing agent such as a stannous salt. The quantity and type of exchange ligand, and amount and type of reducing agent and buffer used may be selected based on the nature of the exchange complex to be formed. The ligand I of the present invention is contained in a second vial, as well as optional additives such as buffers appropriate to adjust the pH to its optimal value.
A single vial kit may be ready for use following addition of the radionuclide ion, such as pertechnetate. A multi-vial kit may be ready for use by addition of the radionuclude ion, such as pertechnetate, to the vial containing exchange ligand and reducing agent, and after waiting an appropriate period of time for formation of a labile complex, the contents of this vial are added to the second vial containing a source of the desired ligand. After a reaction time of about 1 to 60 minutes, the complex of the present invention is formed. It is advantageous that the contents of both vials of this multi-vial kit be lyqphilized. As described for the single vial kit, additional additives may be employed to improve the radiochemical purity and stability of the final product, or to aid in the production of the kit.
Alternatively, the multi-vial kit may comprise the desired ligand in one vial and a source of reducing agent such as stannous ion in a second vial. Pertechnetate may be added to the vial containing ligand, and then the contents of the second vial added to initiate labeling. As above, the quantity and type of ligand, buffer pH and reducing agent may be selected based on the nature of the desired ligand used. Again, it is advantageous that the contents of both vials be lyophilized.
All stereoisomers of the compounds and complexes of the present invention are contemplated herein, whether alone (that is, substantially free of other isomers), in a mixture of certain stereoisomers (for example, as a racemate) or in any other mixture thereof.